Catalytic Nucleic Acids for Treating the Molecular Basis of Disease

During the past decade, antisense technology has emerged as a promising approach
for the treatment of cancer and inflammatory and viral diseases. The antisense
strategy uses short oligonucleotides that bind particular cellular RNAs, leading
to inactivation of the RNAs. The discovery and subsequent development of catalytic
nucleic acids enhanced antisense technology by providing agents that both recognize
and inactivate a target RNA. We used in vitro evolution techniques to develop
novel nucleic acid enzymes that cleave RNA or DNA targets in a sequence-specific
manner. We are investigating the structure and mechanism of these enzymes and
are applying the enzymes to the inactivation of disease-related genes in living
cells.

Rna-Cleaving Dna Enzymes

Several years ago, we used in vitro selection to develop the first example
of a DNA enzyme. Since then, we have produced additional examples, including
DNA enzymes that can be directed to cleave almost any targeted RNA under cellular
conditions. Compared with synthetic RNA enzymes, DNA enzymes are easier to prepare
and are more stable in biological tissues. Our most powerful and versatile DNA
enzyme is the "10-23" motif, which contains a catalytic domain of 15 nucleotides
flanked by substrate-recognition domains of 7­10 nucleotides each (Fig.
1). The catalytic efficiency of this enzyme is the highest of any known nucleic
acid enzyme, limited only by the rate of enzyme-substrate association. The enzyme
binds its RNA substrate through Watson-Crick pairing, enabling us to target different
RNAs simply by altering the sequence of the recognition domains.

We introduced chemical modifications at the 5´ and 3´ ends of the
10-23 enzyme to increase its stability in biological tissues. During the past
year, several laboratories showed that stabilized forms of the enzyme can be
used to inactivate specific target messenger RNAs in cells. Most notably, D.
Snyder and colleagues at the City of Hope Medical Center in Duarte, California,
and K. Taira and colleagues at the Institute of Applied Biochemistry in Tsukuba,
Japan, showed that the enzyme inhibited bcr-abl oncogene expression in bone marrow
cells obtained from patients with leukemia. This inhibition resulted in apoptotic
cell death of the leukemic cells but not the normal cells.

In collaboration with C. Barbas, The Scripps Research Institute, we developed
an RNA-cleaving DNA enzyme that contains 3 essential imidazole-functionalized
deoxyuridine residues in place of thymidine. The in vitro selection scheme was
the same as that used to obtain the 10-23 enzyme, but in this case, the modified
residues, synthesized previously by Barbas and colleagues, were used to provide
added functionality similar to that of the amino acid histidine. The resulting
modified DNA enzyme, the "16.2-11" motif, can be made to cleave RNA substrates
that contain the sequence AUG (Fig. 1). The enzyme has a multiple turnover rate
of greater than 1/min in the presence of micromolar concentrations of zinc.

Structural Studies

We have tried to obtain a high-resolution crystal structure of the 10-23 DNA
enzyme. No DNA enzyme structure has ever been determined. In collaboration with
D. Stout, The Scripps Research Institute, we obtained diffraction-quality crystals
of the enzyme-substrate complex and of 6 corresponding heavy-atom derivatives.
Data were collected at the Stanford Synchrotron Radiation Laboratory, and the
structure of the complex was solved at 3.0-Å resolution.

The structure obtained in the crystal was not the same as that of the molecules
in solution. Instead, an 82-nucleotide complex was formed, consisting of 2 strands
of DNA and 2 strands of RNA. Interestingly, this complex has the structure of
a 4-way junction, analogous to the structure of the Holliday junction that occurs
during genetic recombination. Our structure is the first reported of an all-nucleic-acid
4-way junction, revealing a specifically positioned metal ion that stabilizes
the sharp turn of the DNA backbone at the junction. Efforts continue to obtain
the structure of the 10-23 DNA enzyme in a catalytically relevant conformation,
which would guide further development of the enzyme as a therapeutic agent.